黄条(Seriola aureovittata)MyHC基因克隆及其在早期发育阶段表达研究*

史 宝 孙冉冉, 2 柳学周, 2 徐永江 姜 燕 王 滨 张正荣

史 宝1孙冉冉1, 2柳学周1, 2①徐永江1姜 燕1王 滨1张正荣1

(1. 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071; 2. 大连海洋大学水产与生命学院 大连 116023)

肌纤维体积决定了肌肉的生长发育, 骨骼肌纤维粗肌丝的基本组成单位是肌球蛋白。肌球蛋白是动物体内一种重要的功能性马达蛋白, 在肌肉运动、细胞器运动以及肌肉收缩和信号传导等方面发挥着重要调控作用(Heissler, 2016)。肌球蛋白是由2条肌球蛋白重链(MyHC)和4条肌球蛋白轻链(MyLC)组成。MyHC具有ATP酶活性, 可将其水解为ADP释放能量, 并且含有肌动蛋白结合位点, 为肌肉收缩提供动力从而直接影响鱼类的生长(Knight, 2000; Campion, 2012)。与哺乳动物肌肉生长的模式不同, 鱼类肌纤维生长模式始终存在增生和增粗, 鱼类这种生长现象被称为非限制性生长。研究表明表达量高低影响肌纤维的增生, 与鱼类的非限制性生长有关(Biga, 2009; 陈之航等, 2017)。在翘嘴鳜(),在快长组与慢长组实验鱼中的差异表达提示其在翘嘴鳜胚胎及早期生长发育过程中发挥重要作用(陈之航等, 2017)。对大西洋鲑()的研究表明鱼体特定生长率与表达量的高低有关(Hevrøy, 2006)。对脊尾白虾()的研究发现在仔虾第一天的表达量显著高于胚胎发育的其他时期(王佳佳等, 2019)。

1 材料与方法

1.1 材料及取样

1.2 总RNA的提取与cDNA第一链的合成

1.3 MyHC基因cDNA的RACE克隆

根据克隆得到的核心片段序列及RACE试剂盒要求设计RACE特异引物(表1)。5′RACE反应的操作参照SMARTerTMRACE cDNA Amplification Kit试剂盒方法进行。5′RACE的首次PCR反应体系为25μL: 10×Advantage 2 PCR Buffer 2.5μL, 50×dNTP Mix 2μL, 50×Advantage 2 Polymerase Mix 0.5μL, 5′RACE模板cDNA 1μL, 正向引物UPM 1μL、反向引物MyHC-5′-R1 0.5μL, PCR-Grade Water 17.5μL。PCR反应条件为94°C 30s, 65°C 30s, 72°C 3min, 共25个循环。取首次PCR产物, 加入Tricine-EDTA buffer进行10倍稀释后作为巢式PCR的模板, NUP为正向引物、MyHC-5′-R2为内侧反向引物, 进行目标基因的5′RACE第二次扩增, PCR反应条件同上。3′RACE的操作参照SMARTerTMRACE cDNA Amplification Kit试剂盒方法进行, 3′RACE的PCR反应体系及反应条件与5′RACE相似。RACE产物纯化、克隆和测序方法同上。

1.4 反转录实时荧光定量PCR(qRT-PCR)

Tab.1 Sequence of specific primers used for cloning and quantitative real-time PCR analyses in S. aureovittata

1.5 序列结构及系统进化分析

1.6 数据处理

实验数据用平均值±标准差表示, 并用 Origin 8.0作图。统计分析采用单因素方差分析(One-way ANOVA), 使用SPSS 19.0软件中Duncan's Multiple Range Test 比较基因相对表达水平的差异, 以<0.05作为显著性差异。

2 结果

2.1 MyHC基因序列与结构特征

2.2 MyHC的氨基酸序列比对及系统进化树分析

注：方框表示Src同源区; 下划线表示MYSc-class II; 双下划线表示Myosin tail1

图2 黄条与其他动物MyHC tail1结构域氨基酸序列的多重比较

2.3 MyHC组织表达及早期发育阶段的表达分析

在胚胎发育的各个时期都能检测到的表达, 其中在16细胞期之前表达量较高, 随后显著下降(<0.05); 从原肠胚早期表达量开始再次升高, 胚体下包2/3期表达量显著升高, 至孵化期表达量达到峰值(<0.05)(图5)。在仔稚幼鱼时期,在孵化后的20d之前表达水平较低, 20d后表达量显著升高(<0.05), 在30d表达水平达到峰值, 随后的35d到40d表达水平略有下降但仍保持较高表达趋势至40d, 之后表达量显著下降但仍维持在较高水平(<0.05) (图6)。

图3 基于MyHC氨基酸序列的系统进化树(NJ法, bootstraps=1000)

图4 黄条MyHC在各组织的相对表达量

注：BR. 脑; P. 垂体; L. 肝脏; M. 肌肉; SP. 脾脏; K. 肾脏; GI. 鳃; H. 心脏; ST. 胃; I. 肠; HK. 头肾;

柱上不同小写字母表示显著差异(<0.05)

3 讨论

图5 黄条MyHC在胚胎发育时期的表达

注：1. 受精卵; 2. 2细胞; 3. 4细胞; 4. 8细胞; 5. 16细胞; 6. 32细胞; 7. 多细胞; 8. 桑椹胚; 9. 高囊胚; 10. 低囊胚; 11. 原肠胚早期; 12. 原肠胚中期; 13. 原肠胚末期; 14. 神经胚; 15. 胚体下包1/2; 16. 胚体下包2/3; 17. 胚体全包; 18. 孵化期; 柱上不同小写字母表示显著差异(<0.05); 以受精卵mRNA表达量为标准1

图6 黄条MyHC在仔稚幼鱼发育阶段的表达

注：柱子上不同小写字母表示显著差异 (<0.05); 以孵化后1d表达量为标准1

4 结论

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MOLECULAR CLONING AND mRNA EXPRESSION OFGENE IN THE EARLY DEVELOPMENT OF YELLOWTAIL KINGFISH

SHI Bao1, SUN Ran-Ran1, 2, LIU Xue-Zhou1, 2, XU Yong-Jiang1, JIANG Yan1, WANG Bin1, ZHANG Zheng-Rong1

(1. Laboratory for Marine Fisheries Science and Food Production Processes Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; 2. College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China)

The myosin heavy chain (MyHC) is one of the major structural and contractile proteins of muscle. Study on the early growth and development and gene resources is relatively lacking in yellowtail kingfishTo understand the regulatory roles ofin the early growth and development of, the full-length cDNA sequences ofwas cloned by the RACE technology. The real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the expression patterns ofin different tissues, embryonic development stages, and larval and juvenile stages. The full length of the MyHC cDNA sequence was 6143bp, and the open reading frame was 5811bp, encoding 1936 amino acids. Additionally, common features were found in theof, including MYSc-class II, myosin tail l and SH3 domain. Molecular phylogenetic analysis confirmed thatwas closely related to greater amberjack.was expressed in various tissues, but it had the highest expression in muscle. Developmentally, there was a gradual increase inexpression before the 16-cell stage. And the expression ofincreased significantly in the embryo encircling 2/3 of yolk sac, and reached the peak in the hatching larva (<0.05). In the larval and juvenile stages,was significantly up-regulated on 20 days post hatching (dph), with peak expression occurring on 30 dph and decreased slightly between 35 and 45 dph (<0.05). Theexpression ofshowed a development-stage–specific characteristic. Therefore,plays a regulatory role in the embryo and early developmental stage of.

;; embryonic development; larval and juvenile development; gene cloning; expression analysis

* 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室开放课题, 2017-3A01号; 国家重点研发计划项目, 2018YFD0901204号, 2019YFD0900503号; 国家自然科学基金项目, 31772829号; 中国水产科学研究院院级基本科研业务费-农业部海洋渔业可持续发展重点实验室开放课题资助, 2019HY-XKQ01号; 国家海水鱼产业技术体系项目, CARS-47号。史 宝, 博士, 副研究员, E-mail: shibao@ysfri.ac.cn

柳学周, 研究员, E-mail: liuxz@ysfri.ac.cn

2019-11-06,

2020-01-06

S917.4

10.11693/hyhz20191100205